EFFECT OF CAVITY-CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR-2

Hydrophobic residues in the core of a truncated form of chymotrypsin i nhibitor 2 (CI2) have been mutated in order to measure their contribut ion to the stability of the protein. The free energy of unfolding of w ild-type and mutants was measured by both guanidinium chloride-induced denaturation and differential scanning calorimetry. The two methods g ive results for the changes in free energy on mutation that agree to w ithin 1% or 2%. The average change in the free energy of unfolding (+/ - standard deviation) for an Ile --> Val mutation is 1.2 +/- 0.1 kcal mol-1, for a Val --> Ala mutation 3.4 +/- 1.5 kcal mol-1, and for eith er an Ile --> Ala or a Leu --> Ala mutation 3.6 +/- 0.6 kcal mol-1. Th is gives an average change in the free energy of unfolding for deletin g one methylene group of 1.3 +/- 0.5 kcal mol-1. Two significant corre lations were found between the change in the free energy of unfolding between wild-type and mutant, DELTADELTAG(U-F), and the environment of the mutated residue in the protein. The first is between DELTADELTAG( U-F) and the difference in side-chain solvent-accessible area buried b etween wild-type and mutant (correlation coefficient = 0.81, 10 points ). The second and slightly better correlation was found between DELTAD ELTAG(U-F) and N, the number of methyl/methylene groups within a 6-ang strom radius of the hydrophobic group deleted (correlation coefficient = 0.84, 10 points). The latter correlation is very similar to that fo und previously for barnase, suggesting that this relationship is gener al and applies to the hydrophobic cores of other globular proteins. Th e combined data for C12 and barnase clearly show a better correlation with N (correlation coefficient = 0.87, 30 points) than with the chang e in' the solvent-accessible surface area (correlation coefficient = 0 .82, 30 points). This indicates that the packing density around a part icular residue is important in determining the contribution the residu e makes to protein stability. In one case, Ile --> Val76, a mutation w hich deletes the C(delta1) methyl group of a buried side chain, a surp rising result was obtained. This mutant was found to be more stable th an wild-type by 0.2 +/- 0.1 kcal mol-1. We have solved and analyzed th e crystal structure of this mutant and find that there are small movem ents of side chains in the core, the largest of which, 0.7 angstrom, i s a movement of the side chain that has been mutated. These small move ments in some part compensate for the cavity created by the mutation.